Repair of tympanic membrane using human birth tissue material

ABSTRACT

A tympanic construct fabricated from at least one amniotic membrane, at least one chorionic membrane, or at least one amniotic membrane and at least one chorionic membrane obtained from human birth tissue is provided. Methods of preparing a tympanic construct, methods of repairing tympanic membrane defects and surgical sites, as well as kits for the same are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/194,907, filed Mar. 3, 2014, which claims priority to U.S.Provisional Patent Application No. 61/772,688 filed Mar. 5, 2013, thecontent of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to a tympanic construct fabricatedfrom at least one amniotic membrane, at least one chorionic membrane, orat least one amniotic membrane and at least one chorionic membraneobtained from human birth tissue. The present invention is furtherdirected to methods of preparing a tympanic construct, methods ofrepairing tympanic membrane defects and surgical sites, as well as kitsfor the same.

BACKGROUND OF THE INVENTION

The first component of the middle ear to receive sound waves is thetympanic membrane, also known as the eardrum. Sound waves striking thetympanic membrane are transmitted through a series of bones (e.g.,malleus, incus and stapes) to the cochlea, where the sound waves aresensed and processed.

Tympanic membrane deformities, such as perforations, interfere with thetransmission and perception of sound. Perforations are usually caused bytrauma or infection. Middle ear infections can cause spontaneous rupture(tear) of the eardrum, resulting in a perforation. A hole in thetympanic membrane may also be caused by surgical procedures, e.g., asmall hole may remain in the eardrum after a previously placed pressureequalization tube either falls out or is removed by the physician.

Repair of tympanic membrane perforations is accomplished in a procedureknown as tympanoplasty or myringoplasty. Generally, in tympanoplasty ormyringoplasty, the hole in the tympanic membrane is repaired by means ofa graft. Typical graft materials include natural materials such astemporalis fascia, tragal perichondrium, skin, periosteum, loose overlaytissue, fat, vein tissue, human amniotic membrane, and homologous dura;and non-natural materials such as silastic, paper and teflon sheets.Such typical graft materials used in these procedures, however, maycause further ear infection, loss of hearing, tinnitus, facialparalysis, or hematoma.

Thus, there exists a need for safe and effect materials and techniquesto repair defects of the tympanic membrane.

SUMMARY OF THE INVENTION

The present invention is generally directed to methods and compositionsfor repair of tympanic membranes.

According to one aspect, a tympanic construct is provided. The tympanicconstruct includes at least one amniotic membrane, or at least onechorionic membrane, or at least one amniotic membrane and at least onechorionic membrane. The membrane(s) is/are treated with at least onealcohol composition followed by terminal sterilization to form atympanic construct. According to one embodiment, the alcohol compositioncomprises from about 90% to about 100% ethanol. According to oneembodiment, the terminal sterilization is gamma irradiation or electronbeam irradiation.

According to another aspect, a method of preparing a membrane for atympanic construct is provided. The method includes the steps of:

(a) obtaining amniotic membrane, chorionic membrane, or both amnioticand chorionic membrane from a seronegative, healthy human via Cesareansection or vaginal delivery;

(b) immersing the membrane in a basin containing a sterile salinesolution;

(c) agitating the basin to liberate excess blood and fluids from themembrane;

(d) rinsing the membrane with a sterile saline solution;

(e) covering the membrane with a substrate on both the fetal membraneside and the maternal membrane side;

(f) optionally, immersing the membrane in an alcohol composition;

(g) optionally, rinsing the membrane with a sterile saline solution;

(h) optionally, soaking the membrane in a sterile saline solution;

(i) immersing the membrane in an alcohol composition for a period offrom about 24 hours to about 384 hours;

(j) removing the substrate from both the fetal membrane side and thematernal membrane side;

(k) spreading the membrane on a flat, dry and sterile surface;

(l) allowing the membrane to air dry completely at ambient temperaturefor a period of up to three hours;

(m) cutting the membrane to a predetermined size; and

(n) placing the fetal side of the membrane directly onto a pre-cutsubstrate to form a tympanic construct.

According to one embodiment, the method further includes the steps ofpackaging the tympanic construct in a concentration of ethanol andterminally sterilizing the packaged tympanic construct usingirradiation. According to one embodiment, the method further includesthe steps of packaging the tympanic construct in a dry state andterminally sterilizing the packaged tympanic construct usingirradiation. According to one embodiment, the method further includesthe step of removing the chorionic membrane via blunt dissection anddiscarding the chorionic membrane. According to one embodiment, themethod further includes the step of placing the membrane in sterilesaline solution for a period of up to about five days between steps (a)and (b). In such an embodiment, the sterile saline solution includesfrom about 0.9% to about 20% NaCl. According to one embodiment, thesterile saline solution in step (b) includes from about 0.9% to about20% NaCl. According to one embodiment, the sterile saline solution insteps (d) and (g) each include from about 0.9% to about 20% NaCl, andthe rinse steps (d) and (g) are conducted for a maximum time period offive minutes. According to one embodiment, the sterile saline solutionin step (h) includes from about 0.9% to about 20% NaCl, and the soak instep (h) is conducted for a maximum period of about 35 minutes.According to one embodiment, the alcohol composition in steps (f) and(i) includes from about 90% to about 100% ethanol. According to oneembodiment, the alcohol composition in steps (f) and (i) includes 95.5%ethanol. According to one embodiment, the alcohol composition in steps(f) and (i) includes 100% ethanol.

According to one embodiment, the method further includes the step oftreating the membrane with an oxidizer between steps (a) and (b).According to one embodiment, the oxidizer is hydrogen peroxide and thestep of treating the membrane further includes the steps of:

(a) rinsing the membrane with about 120 ml of sterile isotonic solutionper gram of membrane for a time period of up to about ten minutes;

(b) treating the membrane with about 60 ml of hydrogen peroxide per gramof membrane for a time period of up to about ten minutes; and

(c) rinsing the membrane with about 120 ml of sterile isotonic solutionper gram of membrane for a time period of up to about ten minutes.

According to one aspect, a tympanic construct produced by theaforementioned methods is provided. According to one embodiment, theethanol residual levels determined by gas chromatography are notdetected at the corresponding minimum report limit.

According to one aspect, a method of treating a tympanic membrane defectis provided. The method includes the steps of preparing a tympanicconstruct as provided herein and contacting the tympanic membrane defectwith the tympanic construct. According to one embodiment, the tympanicmembrane defect is a perforation, tear, abrasion, rupture, or puncturein the tympanic membrane. According to one embodiment, the methodfurther includes the step of hydrating the tympanic construct prior toplacement.

According to one aspect, a method of treating a tympanic membranesurgical site is provided. The method includes the steps of preparing atympanic construct as provided herein and contacting the tympanicmembrane surgical site with the tympanic construct.

According to another aspect, a kit for use by a surgical professional isprovided. According to one embodiment, the kit includes one or morepackaged and sterilized tympanic constructs as provided herein. The kitmay further include at least one set of instructions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of preparing a tympanic construct accordingto one embodiment.

FIG. 2 illustrates a method of preparing a tympanic construct accordingto one embodiment.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure will now be described more fully hereinafter withreference to exemplary embodiments thereof. These exemplary embodimentsare described so that this disclosure will be thorough and complete, andwill fully convey the scope of the disclosure to those skilled in theart. Indeed, the present disclosure may be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure will satisfy applicable legal requirements. As used in thespecification, and in the appended claims, the singular forms “a”, “an”,“the”, include plural referents unless the context clearly dictatesotherwise.

As used herein, and in the appended claims, the term “construct” refersto a patch, graft, or other material embodiment suitable for contactingthe tympanic membrane or surrounding tissue.

As used herein, and in the appended claims, the terms “optional” or“optionally” mean that the subsequently described event or circumstancecan or cannot occur. For example, the phrase “optionally soaking themembrane” means that the soaking step may or may not be performed.

As used herein, and in the appended claims, the term “human birthtissue” includes, but is not limited to, elements of the placental organsuch as, for example, the placental globe, umbilical cord, umbilicalcord blood, amniotic membrane, chorionic membrane, and other placentalgelatins, fluids, cells and extracellular material obtained from aseronegative, healthy human.

The use of amniotic membrane, chorionic membrane, or a combination ofamniotic and chorionic membranes obtained from human birth tissue toform a tympanic construct are generally provided. The tympanic constructmay act to repair a tympanic membrane defect as well as reducepost-operative scar tissue formation, reduce pain, reduce inflammation,and, generally, aid in the healing cascade. Methods for asepticallyprocessing intact amnion and/or chorion membranes to produce a materialthat may be used to prepare a tympanic construct are also provided.

The amniotic and chorionic membranes may be utilized alone or in variouscombinations or layers to form the tympanic construct. The term“membrane” refers to an amniotic membrane, a chorionic membrane, or botha chorionic and an amniotic membrane. According to one embodiment, thetympanic construct includes one or more layers including an amnioticmembrane. According to another embodiment, the tympanic constructincludes one or more layers that include a chorionic membrane. Accordingto yet another embodiment, the tympanic construct includes one or morelayers of an amniotic membrane and one or more layers of a chorionicmembrane.

According to one embodiment, the tympanic construct includes at leastone amniotic membrane, or at least one chorionic membrane, or at leastone amniotic membrane and at least one chorionic membrane. In such anembodiment, the membrane(s) is/are not cross-linked. The membrane(s)is/are treated with at least one alcohol composition that includestypically from about 90% to about 100% ethanol. The resultingmembrane(s) is/are terminally sterilized to form a tympanic construct.

To obtain human birth tissue material, potential human birth tissuedonors providing informed consent are pre-screened during an examinationof pre-natal medical records and blood test results. A comprehensivemedical history and behavior risk assessment is obtained from the donorprior to donation incorporating U.S. Public Health Service guidelines.Discussions with the physician(s) and/or the donor mother are conductedto identify circumstances that may lead to the exclusion of the donor ordonated tissue. Additionally, a physical exam is performed on the donorto determine whether there is evidence of high risk behavior orinfection and to determine the overall general health of the donor.

Infectious disease testing of donor blood specimens is performed foreach tissue donor on a specimen collected at the time of donation orwithin seven days prior to or after donation. Exemplary infectiousdisease testing includes, but is not limited to, antibodies to the humanimmunodeficiency virus, type 1 and type 2 (anti-HIV-1 and anti-HIV-2);nucleic acid test (NAT) for HIV-1; hepatitis B surface antigen (HBsAg);total antibodies to hepatitis B core antigen (anti-HBc—total, meaningIgG and IgM); antibodies to the hepatitis C virus (anti-HCV); NAT forHCV; antibodies to human T-lymphotrophic virus type I and type II(anti-HTLV-I and anti-HTLV-II); and syphilis (a non-treponemal ortreponemal-specific assay may be performed).

Human birth tissue is preferably recovered from a full-term asepticCesarean delivery of a newborn. Alternatively, human birth tissue isrecovered from a full-term vaginal delivery of a newborn. The placentalorgan, including the placental globe, umbilical cord, associatedmembranes (chorionic membrane and amniotic membrane), and othergelatins, fluids, cells and extracellular matrix can be recovered from aseronegative, healthy human after the newborn is removed. The placentalglobe, umbilical cord, and other gelatins, fluids, cells andextracellular matrix can be removed and discarded.

The membrane(s) giving rise to the tympanic construct as describedherein may be produced by processing human birth tissue according to thesteps provided herein. Processing does not change the physicalproperties of the resulting membrane to yield the membrane tissueunacceptable for clinical use. Instruments, solutions, and suppliescoming into contact with tissue during the processing of the placentaltissue are sterile. All surfaces coming in contact with tissue intendedfor transplant are either sterile or draped using aseptic technique.

Throughout processing, the orientation of the particular membrane isidentified to ensure that in use, the correct side of the membrane isplaced on or around the tympanic membrane. Either the fetal side or thematernal side of the membrane may be placed on or around the tympanicmembrane, depending upon the specific use/procedure and the tympanicconstruct composition (i.e., the type of membrane(s) used and the numberof membrane layers).

According to the embodiment as illustrated in FIG. 1 or FIG. 2, thetympanic construct is prepared by first obtaining amniotic membrane,chorionic membrane, or both amniotic and chorionic membrane from aseronegative, healthy human via cesarean section or vaginal delivery asdescribed herein. In particular embodiments where only the amnioticmembrane is chosen for further processing, the chorionic membrane can beremoved by blunt dissection. For example, the chorionic membrane may beremoved by applying finger pressure and sliding it off of the amnioticmembrane using as little pressure as possible to avoid tearing of theamnion. The chorionic membrane and any excess tissue can be discarded.

The recovered amniotic membrane, chorionic membrane, or both amnioticand chorionic membrane may be initially stored in a sterile salinesolution at a temperature between about 1° C. to about 10° C. for aperiod of up to about five days prior to further processing. Accordingto one embodiment, the sterile saline solution comprises from about 0.9%to about 20% NaCl, preferably 15% NaCl.

Optionally, the membrane(s) may be treated with an oxidizer. In oneembodiment, the oxidizer is hydrogen peroxide, which is also used as asterilant and to enhance the solubilization of lipids. Such a treatmentprocess includes the steps of:

(a) rinsing the membrane with 120 ml of sterile isotonic solution pergram of membrane for a period of up to about ten minutes;

(b) treating the membrane with 60 ml of hydrogen peroxide per gram ofmembrane for a period of up to about ten minutes; and

(c) rinsing the membrane with 120 ml of sterile isotonic solution pergram of membrane for a period of up to about ten minutes.

The membrane is then immersed in a basin containing a sterile salinesolution. According to one embodiment, the sterile saline solutionincludes typically from about 0.9% to about 20% NaCl.

Excess blood and fluids may be liberated from the membrane by gentlystirring or swirling the fluid in a circular motion in the basin or byplacing the basin on a shaker. The membrane can then be rinsed with asterile saline solution. In one embodiment, the sterile saline solutionincludes NaCl in a concentration range of about 0.9% to about 20%. Inone embodiment, the membrane may be rinsed in bowls or trays ofsufficient size to allow the membrane to be spread out to improve therinse coverage. Sufficient saline solution is utilized to ensure thatthe membrane is completely immersed. The saline is then decanted into adiscard basin.

Multiple saline rinse cycles may be performed. In one embodiment, themembrane is rinsed for two or more separate rinse cycles, with eachrinse cycle lasting for a maximum of five minutes. The membrane iscovered with a substrate on both the fetal membrane side and thematernal membrane side. Appropriate substrates include, but are notlimited to, sterile mesh or polymer mesh of adequate size and shape forcovering each side of the membrane.

According to the embodiment as illustrated in FIG. 2, the membrane mayoptionally be immersed in an alcohol composition from about 90% to about100% ethanol (see FIG. 2—referred to as “first alcohol composition”). Incertain embodiments, the alcohol composition includes about 95.5%ethanol. In a preferred embodiment, the alcohol composition includesabout 100% ethanol.

As illustrated in the embodiment of FIG. 2, the membrane is thenoptionally rinsed with a sterile saline solution. Alternatively, themembrane is rinsed multiple times with a sterile saline solution.According to one embodiment, the sterile saline solution includestypically from about 0.9% to about 20% of NaCl. According to oneembodiment, the rinse step is conducted for a maximum time period offive minutes. The membrane can then be optionally soaked in a sterilesaline solution. According to one embodiment, the sterile salinesolution includes typically from about 0.9% to about 20% of NaCl.According to one embodiment, soaking is conducted for a maximum periodof about 35 minutes.

As illustrated in each of the embodiments of FIGS. 1 and 2, the membraneis immersed in an alcohol composition for a period of typically fromabout 24 hours to about 384 hours. The alcohol composition includesabout 90% to about 100% ethanol. In certain embodiments, the alcoholcomposition includes about 95.5% ethanol. In a preferred embodiment, thealcohol composition includes about 100% ethanol. Treatment of themembrane within a particular alcohol concentration range for theparticular timeframe at this step in the process has yielded unexpectedresults related to the handling characteristics. One of ordinary skillin the art appreciates the difficulty of handling and manipulatingamniotic and chorionic tissue when applied to a specific site.Specifically, existing amniotic and chorionic grafts are difficult toplace over a specific site, particularly because these grafts fold backover on themselves (“wrinkling”), rendering proper placement andpositioning of the graft at the wound site very challenging. Whentreated with the aforementioned alcohol composition for the particulartimeframe, the resulting tympanic construct exhibits improved handlingcharacteristics in that it does not “wrinkle” and allows for easyplacement at the chosen site. Additionally, the membrane is uniquelyprocessed to allow adhesion to the surrounding tympanic membrane tissuewithout the aid of support materials, tissue glue/adhesives or sutures.Furthermore, the alcohol treatment is multi-functional, providing ameans of sterilization, preservation, and chemical dehydration for themembrane, in addition to serving as a radioprotectant for the membraneprior to terminal irradiation.

The substrate can then be removed from both the fetal membrane side andthe maternal membrane side. The membrane can then be spread on a flat,dry and sterile surface. The membrane is then allowed to air drycompletely at ambient temperature for a period of up to typically aboutthree hours. The membrane can then be cut to the desired size,optionally covered with a substrate, and subsequently packaged. Thetympanic construct can be cut into patches of any desired size for aparticular application by a rotary type cutting tool. A grooved orsimilarly indicated cutting board may be used to aid in cutting astraight and correctly sized covering. In another embodiment, thetympanic construct is cut by free hand using a scalpel and ruler toachieve the desired size. The fetal side of the membrane can then beplaced directly onto a pre-cut substrate to form a tympanic construct.Suitable substrates include, for example, a gauze or synthetic mesh. Thetympanic construct can be packaged in a dry state. Alternately, thetympanic construct can be packaged in sterile water, crystalloids, oranother sterilizing, preserving or storage agent, including ethanol. Thecovering can be removed after the opposing side has been applied to thesurgical site. The packaging and covering as disclosed herein canfacilitate the handling of the tympanic construct, namely maintainingand identifying the orientation of the fetal and maternal side of thetympanic construct for the user. The packaging may also promote storageof the tympanic construct.

The packaged tympanic construct can be terminally sterilized usingirradiation. In one embodiment, an electron beam irradiation is appliedin an amount up to about 45 kGy. The sterilized tympanic construct maybe stored for up to typically about two years from the date ofprocessing. In one embodiment, the tympanic construct may be storedunder proper conditions for as much as about five years followingprocessing. The sterilized tympanic construct may be stored in anycontainer suitable for long-term storage. Preferably, the sterilizedtympanic construct is stored in a sterile double peel-pouch package.

The tympanic construct as described herein may be of various sizes,thicknesses, and shapes. The tympanic construct is preferably ofsufficient size and shape to be applied onto or around a tympanicmembrane defect. The tympanic construct may be any shape or conformationthat facilitates the treatment of a portion of the existing tympanicmembrane. In certain embodiments, the tympanic construct may be shapedas a square, rectangular, circular or oval, or may be cut to conformgenerally to the shape of the defect. The thickness of the tympanicconstruct may vary depending on application, the type of membrane andthe number of membrane layers. Typically, the tympanic construct is fromabout 0.01 μm to about 200 μm thick.

A method of treating a tympanic membrane defect is also provided. Beforeattempting any correction of the defect, a hearing test can beperformed, and the patient can be evaluated for Eustachian tubefunction, as partial or complete loss of Eustachian tube function canexacerbate a tympanic membrane defect (e.g., puncture) and interferewith the adherence of a construct to the tympanic membrane. The methodincludes the steps of preparing a tympanic construct as provided hereinand placing the tympanic construct on or around a tympanic membranedefect. According to one embodiment, the repair of the tympanic membraneis a tympanoplasty. According to another embodiment, the repair of thetympanic membrane is a myringoplasty. According to either embodiment,the otolaryngologist may approach repair of a tympanic membrane defecteither through the auditory canal (trans-canal approach), or via apost-auricular incision followed by folding the ear forward to exposethe tympanic membrane (post-auricular approach).

The tympanic membrane defect can be any perforation, tear, abrasion,rupture, puncture wound, or other trauma. Such a defect may be causedaccidentally, by trauma, by infection, or may be caused deliberately(e.g., from insertion of one or more tubes allowing drainage of fluidsin the middle ear past the tympanic membrane and out the auditory canal(e.g., perforation(s) to allow a myringotomy tube installation, or aperforation caused by surgical removal of diseased or damaged tissue).The defect may be acute, or the defect may be chronic (e.g., inexistence for two months or more). In certain embodiments, the defectmay be a deformity that has arisen as the result of a disease orinfection or was present at birth.

A method of treating a surgical site is also provided. The methodincludes the steps of preparing a tympanic construct as provided hereinand placing the tympanic construct on or around a portion of thetympanic membrane subject to a surgical procedure.

If desired, the one or more membranes forming the tympanic constructutilized in the methods provided herein may be treated, coated orimpregnated with one or more of a variety of optional components to aidin defect resolution, healing, and recovery. Exemplary optionalcomponents include, but are not limited to, antibiotics,anti-inflammatory agents, anti-viral agents, growth factors,antiproliferative agents, cytokines, antihistamines, pain medications,biocides, cellular attractant and scaffolding reagents, wound healingagents or sealants, nutritional agents (e.g., vitamins), hormones,alkylating agents, immunomodulatory agents (e.g., steroids), and/orother specialized proteins or small molecules. The one or more membranesforming the tympanic construct may be combined with a substrate (sterilegauze, sterile polymer material, or other tissue or biomaterial) toincrease the strength of the tympanic construct.

If desired, the one or more membranes forming the tympanic construct maybe utilized with at least one composition or device for delivering,fastening or fixing the tympanic construct on or around a tympanicmembrane defect. Exemplary compositions include, but are not limited to,tissue glue or tissue adhesive, fibrin glue, fibrinogen glue, hydrogeltissue glue, chondroitin sulfate aldehyde, or natural proteins.Exemplary devices include, but are not limited to, sutures or forceps.

A kit for use by a surgical professional is also provided. According toone embodiment, the kit includes one or more packaged and sterilizedtympanic constructs as provided herein. The kit may further include atleast one set of instructions. The kit may further include a containeradapted to accommodate the aforementioned components while preservingthe tympanic construct as per applicable Food and Drug Administrationguidelines.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation of the invention. Modifications will be obvious to thoseskilled in the art, and all modifications that do not depart from thespirit of the invention are intended to be included with the scope ofthe appended claims.

Having generally described the present invention, a furtherunderstanding can be obtained by reference to the examples providedherein for purposes of illustration only and are not intended to belimiting.

Example 1

Three representative samples of final product for each of threeproduction lots manufactured according to the methods of FIG. 1 weretested for residual ethanol by gas chromatography, analytical method EPA8260B, CAS No. 64-17-5. Samples were sent to Nelson Laboratories, Inc.,6280 South Redwood Road Salt Lake City, Utah 84123, a GLP qualifiedmicrobiology laboratory registered with the FDA and third-partyaccredited to ISO 17025 standards.

The three samples submitted for testing from production lot #5 (2 cm×3cm; 2 cm×3 cm; and 1.5 cm×2 cm) included amniotic membranes that hadbeen immersed in an alcohol composition comprising 95.5% ethanol for aperiod of 110 hours. The three samples submitted for testing fromproduction lot #6 (2 cm×3 cm each) included amniotic membranes that hadbeen immersed in an alcohol composition comprising 95.5% ethanol for aperiod of 25.5 hours. The three samples submitted for testing fromproduction lot #7 (2 cm×3 cm each) included amniotic membranes that hadbeen immersed in an alcohol composition comprising 95.5% ethanol for aperiod of 24.9 hours.

Zero headspace extraction was performed with double deionized water asthe vehicle extractant. Extraction vessels were tumbled during theentire extraction process. For each of the three production lots, thesamples were pooled, and one test article was extracted with a weight of0.05 g and fluid amount of 50 ml. The starting extraction temperaturewas 22° C. and the ending extraction temperature was 23° C. Theextractions lasted twenty-four hours. All sample extract solutions wereobserved to be clear and free of particulates. At the end of theextraction period, all test articles were observed to be intact with noobservable degradation. Extracts were maintained at room temperature andwere not filtered prior to analysis. The vehicle solution was introducedinto a purge and trap unit suitable for gas chromatography-massspectrometry analysis. Control blanks contained no compounds of interestat the reported detection limits. Low level calibration standards wereanalyzed at the detection levels, and standard percent recoveries werewithin acceptable method limits. No analytical interferences wereobserved. All instrument calibration results were within methodrequirements through all portions of the analysis.

The certificates of analyses for production lots #5, #6 and #7 indicatedno detectable amounts of ethanol at the minimum reporting limit (0.5mg/L). The results are summarized in Table 3 below.

TABLE 3 Ethanol Determination for Production Lots #5, #6 and #7 TotalStarting Ending Production Time in Weight of Volume ExtractionExtraction Duration of Sample Lot Ethanol Sample of Fluid TemperatureTemperature Extraction Results 5 110.0  0.05 g 50 mL 22° C. 23° C. 24Hours ND* Hours 6 25.5 0.05 g 50 mL 22° C. 23° C. 24 Hours ND* Hours 724.9 0.05 g 50 mL 22° C. 23° C. 24 Hours ND* Hours *ND = Not Detected atthe Minimum Reporting Limit (0.5 mg/L)

I claim:
 1. A method of preparing a membrane for a tympanic construct,comprising the steps of: (a) obtaining amniotic membrane, chorionicmembrane, or both amniotic and chorionic membrane from a seronegative,healthy human via Cesarean section or vaginal delivery; (b) immersingthe membrane in a basin containing a sterile saline solution; (c)agitating the basin to liberate excess blood and fluids from themembrane; (d) rinsing the membrane with a sterile saline solution; (e)covering the membrane with a substrate on both the fetal membrane sideand the maternal membrane side; (f) immersing the membrane in an alcoholcomposition of 90% to 100% ethanol for a period of from 24 hours to 384hours; (g) removing the substrate from both the fetal membrane side andthe maternal membrane side; (h) spreading the membrane on a flat, dryand sterile surface; (i) allowing the membrane to air dry completely atambient temperature for a period of up to three hours; (j) cutting themembrane to a predetermined size; and (k) placing the fetal side of themembrane directly onto a pre-cut substrate to form a tympanic construct.2. The method of claim 1, further comprising the steps of: (o) packagingthe tympanic construct in a concentration of ethanol; and (p) terminallysterilizing the packaged tympanic construct using irradiation.
 3. Themethod of claim 1, further comprising the steps of: (o) packaging thetympanic construct in a dry state; and (p) terminally sterilizing thepackaged tympanic construct using irradiation.
 4. The method of claim 1,wherein step (a) further comprises the step of removing the chorionicmembrane via blunt dissection and discarding the chorionic membrane. 5.The method of claim 1, further comprising the step of placing themembrane in sterile saline solution for a period of up to five daysbetween steps (a) and (b), the sterile saline solution comprising from0.9% to 20% NaCl.
 6. The method of claim 1, wherein the sterile salinesolution in step (b) comprises from 0.9% to 20% NaCl.
 7. The method ofclaim 1, wherein the sterile saline solution in steps (d) and (g) eachcomprise from 0.9% to 20% NaCl, and wherein rinse steps (d) and (g) areconducted for a maximum time period of five minutes.
 8. The method ofclaim 1, wherein the sterile saline solution in step (h) comprises from0.9% to 20% NaCl, and wherein the soak in step (h) is conducted for amaximum period of 35 minutes.
 9. The method of claim 1, wherein thealcohol composition in steps (f) and (i) comprises 95.5% ethanol. 10.The method of claim 1, wherein the alcohol composition in steps (f) and(i) comprises 100% ethanol.
 11. A tympanic construct produced by themethod of claim
 1. 12. The tympanic construct of claim 11, wherein theethanol residual levels determined by gas chromatography are notdetected at the corresponding minimum report limit.